Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis
Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis
复制标题
磷嗪霉素生物合成中硝基形成酶的表征
DOI：
10.1021/acs.biochem.1c00512
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发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
Sobrado, Pablo
中科院分区：
文献类型：
--
作者：
Valentino, Hannah;Sobrado, Pablo
N-羟基化单加氧酶（NMOs）是羟基化氮原子的黄素依赖性酶的亚类。最近，已经鉴定出在一个底物分子上进行多个反应的独特的NMO。磷嗪霉素M（FzmM）是一种这样的NMO，在某些链霉菌的磷嗪霉素生物合成途径中从天冬氨酸（Asp）形成硝基琥珀酸。这项工作详细介绍了FzmM的生化和动力学分析。稳态动力学研究表明，FzmM与Asp发生偶联反应（kcat，3.0 ± 0.01 s-1），生成硝基琥珀酸酯，在FzmL作用下，生成反丁烯二酸酯和亚硝酸酯. FzmM对NADPH的kcat/Km值比对NADH的kcat/Km值高70倍，并且具有窄的最佳pH范围（7.5-8.0）。与kredis限速的其他NMOs相反，FzmM与NADPH的kredis非常快（4 °C时为50 ± 0.01 s-1）。NADPH结合的Kd值约为400 μM，氢化物转移以pro-R立体化学发生。在不存在Asp的情况下，FzmM的氧化显示出在λ 370 nm处具有肩峰的光谱，这与C（4a）-过氧化氢黄素中间体的形成一致，其以比kcat慢100倍的速率衰变成氧化黄素和过氧化氢。该反应在Asp存在下增强，具有比thekcat稍快的kox，表明黄素脱水或Asp氧化是部分速率限制的。FzmM到NMOs的多序列分析鉴定了涉及黄素结合但不涉及NADPH的保守残基。对相关单加氧酶的额外序列分析表明，FzmM共享在其他NMO中不存在的序列基序。
N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in someStreptomycessp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s–1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higherkcat/KMvalue for NADPH compared to NADH and has a narrow optimal pH range (7.5–8.0). Contrary to other NMOs where thekredis rate-limiting, FzmM exhibits a very fastkred(50 ± 0.01 s–1at 4 °C) with NADPH. NADPH binds at aKDvalue of ∼400 μM, and hydride transfer occurs withpro-Rstereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than thekcat. This reaction is enhanced in the presence of Asp with a slightly fasterkoxthan thekcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.